1. Field of the Invention
The present invention relates to a method of inhibiting protein kinase C function and to compounds and compositions suitable for use in such a method.
2. Background Information
In mouse skin, phorbol esters exert tumor promoting, inflammatory, and hyperplastic activity. These responses are thought to be mediated via the major phorbol ester receptor, protein kinase C (Nishizuka, Nature, 308: 693-698, 1984; Nishizuka, Science, 233: 305-312, 1986; Nishizuka, Nature, 334: 661-665, 1988; Blumberg et al, In: T. J. Slaga (ed.) Mechanisms of Tumor Promotion, Tumor Promotion and Carcinogenesis In Vitro, vol. 3, pp. 143-184, Boca Raton, Fla.: CRC Press, 1984).
Using [H.sup.3 ]PDBu, Blumberg and co-workers characterized specific, high-affinity phorbol ester binding sites in particulate preparations from mouse skin and mouse epidermis. The measurements yielded curved Scatchard plots, consistent with receptor heterogeneity (Dunn et al, Cancer Res., 43: 4632-4637, 1983). These were the first results suggesting that the phorbol ester receptors, subsequently identified as PKC, might represent a family of isoforms differing in structure-activity relations. It is now known that protein kinase C indeed consists of at least 9 isozymes (Blumberg, Cancer Res., 48: 1-8, 1988; Nishizuka, Cancer 63: 1982-1903, 1989). The binding characteristics of only the alpha, beta, and gamma isozymes have been investigated in detail so far. These three isozymes appear quite similar for recognizing the phorbol esters, although differences in interaction with unsaturated fatty acids have been noted (Sekiguchi et al, Biochem. Biophys. Res. Commun., 145: 797-802, 1987; Akita et al, J. Biol. Chem., 265: 354-362, 1990).
Despite the lack of biochemical understanding, whole animal analysis argues strongly for heterogeneity in response to the phorbol esters. A decade ago, Hecker and co-workers demonstrated that 12-deoxyphorbol 13-phenylacetate, -isobutyrate, or -angelate were inflammatory but either not promoting or weakly promoting (Hergenhahn et al, J. Cancer Res. Clin. Oncol., 104: 31-39, 1982). Similar behavior was noted for phorbol esters with unsaturated side chains (Furstenberger et al, Planta Medica, 22: 241-266, 1972). The groups of Slaga (Proc. Natl. Acad. Sci. USA, 77: 3659-3663, 1980; Slaga Environ. Health Perspect., 50: 3-14, 1983) and Marks (Proc. Natl. Acad. Sci. USA, 78: 7722-7726, 1981) showed that tumor promotion could be subdivided into distinct stages differing in structure-activity requirements; mezerein and 12-O-retinoylphorbol 13-acetate, although only weakly promoting themselves, were effective if preceded by one or more applications of phorbol 12-myristate 13-acetate (PMA).
In all of the above cases, the compounds induce in cultured cells essentially the complete spectrum of phorbol ester responses. This is not so for the bryostatins, a structurally distinct class of protein kinase C activators. The bryostatins induce only some of the responses seen for the phorbol esters; when co-applied with the phorbol esters, the bryostatins block those responses which they themselves do not induce (Blumberg, Cancer Res., 48: 1-8, 1988). In mouse keratinocytes, the bryostatins fail to induce markers of differentiation (Sako et al, Cancer Res., 47: 5445-5450, 1987). In mouse skin, the bryostatins are inactive as first stage promoters (Gschwendt et al, Carcinogenesis, 9: 555-562, 1988), very weak as second stage promoters or as complete promoters (Hennings et al, Carcinogenesis, 8: 1343-1346, 1987), strong inhibitors of first stage promotion (Gschwendt et al, Carcinogenesis, 9: 555-562, 1988), and modest inhibitors of complete promotion (Hennings et al, Carcinogenesis, 8: 1343-1346, 1987).
One other report of protein kinase C activators which inhibit tumor promotion remains difficult to evaluate. Schmidt and Hecker (In: E. Hecker, N. E. Fusenig, W. Kunz, F. Marks and H. W. Thielmann (eds.) Cocarcinogenesis and Biological Effects of Tumor Promoters, pp. 57-63. New York: Raven Press, 1982) reported that co-application of PMA and a 4- to 8-fold higher dose of phorbol 12,13-diacetate, -dibutyrate, -dipropionate, or -dibenzoate completely blocked promotion in NMRI mice. This was clearly not seen for phorbol 12,13-diacetate in SENCAR mice (Slaga et al, Proc. Natl. Acad. Sci. USA, 77: 3659-3663, 1980), and phorbol 12,13-dibutyrate and phorbol 12-13-dibenzoate are themselves tumor promoting (Scribner et al, Europ. J. Cancer, 8: 617-621, 1972; Baird et al, Cancer Res., 31: 1074-1079, 1971). In the same paper, Schmidt and Hecker also reported that a single co-treatment had no effect on thymidine incorporation or hyperplasia in NMRI mouse skin and that, after eight cotreatments, there was only a slightly less intense hyperplasia than after eight treatments with PMA alone.
Applicants have characterized the biological effects of 12-deoxyphorbol 13-acetate (prostratin). Specifically, Applicants have examined the effect of single or multiple pretreatments of mouse skin with prostratin on the subsequent response to PMA. The results indicate that prostratin pretreatment inhibits the response to PMA, with varying characteristics for different responses. Prostratin thus represents a novel class of physiological antagonist for protein kinase C.